In chromosomal DNA of a higher eukaryote such as human, cytosine in a CpG site comprising a CG dinucleotide sequence is methylated in some cases. This methylation of cytosine in a CpG site functions as a mechanism for suppressing expression of genes. For example, a region containing a large amount of CpG sites is present in a promoter region of a gene, and on-off of transcription from DNA of the gene is controlled by the presence or absence of methylation of cytosine in this promoter region of a gene.
Control of gene expression by DNA methylation plays an important role in events such as early embryo development, tissue-specific gene expression, gene imprinting, inactivation of X chromosome, stabilization of chromosome, and typing of DNA replication. In addition, it has been reported that DNA methylation may be highly involved in diseases such as cancer.
As a method for analyzing DNA methylation described above, methylation-specific PCR method or bisulfite sequencing method is generally used. In these methods, a treatment of converting cytosine that is not methylated (unmethylated cytosine) in DNA to be analyzed into another base (unmethylated cytosine conversion treatment) is carried out by using bisulfite that is a reagent for converting unmethylated cytosine into another base (unmethylated cytosine conversion agent). In the Methylation-specific PCR method, DNA methylation is analyzed by carrying out a polymerase chain reaction of each of a primer set in the case where cytosine is converted into another base and a primer set in the case where cytosine is not converted into another base using DNA after the unmethylated cytosine conversion treatment and examining the presence or absence of amplification product. In addition, in the bisulfite sequencing method, DNA methylation is analyzed by determining a nucleotide sequence of DNA after the unmethylated cytosine conversion treatment.
Therefore, by the methylation-specific PCR method and the bisulfite sequencing method, methylated DNA cannot be accurately detected unless an unmethylated cytosine conversion treatment is properly carried out. Therefore, determination of whether or not an unmethylated cytosine conversion treatment is properly carried out is required to carry out the accurate detection of methylated DNA.
Incidentally, Patent Publication 1 discloses, as a primer for confirmation of whether or not bisulfite treatment is properly carried out, a primer that is designed based on a region not containing a CG sequence and has a nucleotide sequence in which cytosine is converted into thymine in the nucleotide sequence.
[Patent Publication 1] Japanese Patent Laid-Open No. 2005-58217